Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (1): 170-181.doi: 10.3864/j.issn.0578-1752.2025.01.013

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Effects of Alcalase Hydrolysis on the Structure, Aggregation Behavior and Gelling Properties of Quinoa Protein Isolate

FENG Xiao(), WEI JianFeng(), FU LiXiao, WU ChaoSheng, YANG YuLing, TANG XiaoZhi*()   

  1. College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety of Jiangsu Province/Key Laboratory for Quality Safety Control and Deep Processing of Cereals and Oils, Nanjing 210023
  • Received:2024-05-06 Accepted:2024-11-27 Online:2025-01-01 Published:2025-01-07
  • Contact: TANG XiaoZhi

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Abstract:

【Objective】This research studied the effects of alcalase hydrolysis on the structure, physicochemical properties and aggregation behavior of quinoa protein isolate (QPI), and explored its effects on the gelling properties of QPI. 【Method】QPI was extracted by alkaline extraction and acid precipitation method at 4 ℃. Alcalase with different enzyme-substrate ratios was added to the QPI solution to hydrolyze the protein. Thereafter, the changes of the composition, particle size, Zeta potential, solubility, and surface hydrophobicity (S0-ANS) of QPI were analyzed, and the correlation between these changes and the Th T fluorescence intensity and morphology of protein thermal aggregates were discussed. Furthermore, the concentration of QPI dispersion was elevated to form heat-induced QPI gels. The correlation among alcalase hydrolysis, aggregation behavior and gelling properties of QPI was discussed through the analysis of the microstructure, texture and protein secondary structure of QPI gels. Meanwhile, the reasons for the changes in the gelling properties of QPI induced by alcalase hydrolysis were revealed.【Result】With the increase of enzyme substrate ratio (E/S), the protein particle size of QPI dispersion (2%, w/v) gradually decreased, and its electronegativity increased first and then decreased. Meanwhile, the surface hydrophobicity of QPI gradually increased as the E/S increased. At the same time, the Th T fluorescence intensity of QPI aggregates showed an upward trend as E/S rose from 0 to 0.08%, and then declined when the E/S further increased to 0.14%. Through TEM observation, short fibrils (145-306 nm) and long fibrils (217-406 nm) were formed when E/S was 0.05% and 0.08%, respectively. However, with the further increase of E/S ratio, the length of fibrils became shorter, and more amorphous aggregates appeared. In addition, it was found that the aggregation behavior of QPI had a significant effect on its gelling properties (P<0.05). QPI gels showed enhanced hardness, higher storage modulus and a denser network structure, when fibrillar aggregation was dominant. Compared with short fibrils, long fibrils exerted a more significant effect to improve the gelling properties. Furthermore, alcalase showed a significant effect on the protein secondary structure of QPI gels, and the contents of β-sheet and random coil rose first and then decreased with the increase of E/S.【Conclusion】The limited alcalase hydrolysis promoted QPI to form ordered fibrillar aggregates, and further improved its gelling properties. When the E/S was 0.08%, QPI formed the longest fibrils, and QPI gels exhibited the highest hardness as well as the most favorable viscoelastic properties. Meanwhile, the protein secondary structure of QPI gels was ordered, and their microstructure was dense. However, the higher degree of hydrolysis was unfavorable to form fibrillar aggregates and failed to improve the gelling properties. Therefore, limited alcalase hydrolysis could significantly improve the weak gelling properties of QPI through fibrillation.

Key words: quinoa protein isolate (QPI), alcalase, limited hydrolysis, fibrillar aggregates, gel network, protein secondary structure

Fig. 1

The SDS-PAGE image of quinoa protein isolate under different enzyme-substrate ratio treatments M: Marker; 1-5 are Control group, enzyme-substrate ratio (E/S, w/w) is 0.05%, 0.08%, 0.11% and 0.14%, respectively"

Table 1

The protein composition of QPI under different enzyme-substrate ratio treatments"

酶底比
Enzyme substrate ratio (%)		 条带强度Band intensity (%)
非还原条件 Non-reducing condition 还原条件 Reducing condition
7S & 11S 2S 7S AS BS 2S
对照 Control 52.59 19.84 19.80 28.31 31.70 19.32
0.05 48.13 18.92 14.14 23.81 26.53 17.64
0.08 46.01 16.19 13.94 22.41 26.17 15.23
0.11 41.25 15.05 13.12 23.45 23.27 13.93
0.14 37.64 16.18 9.25 21.37 22.35 14.23

Table 2

Average particle size, PDI, and zeta potential of QPI"

酶底比
Enzyme substrate ratio (%)		 粒径
Particle size (nm)		 分散性指数
PDI		 Zeta电位
Zeta potential (mV)
对照 Control 1163.60±305.39a 0.59±0.15a -11.87±0.35a
0.05 904.37±222.62ab 0.51±0.06a -13.17±0.37b
0.08 603.13±107.06b 0.39±0.11a -14.93±0.12d
0.11 571.27±40.81b 0.53±0.35a -14.73±0.67cd
0.14 545.60±82.69b 0.48±0.64a -13.83±0.81bc

Fig. 2

Effects of different enzyme-substrate ratios on the solubility and surface hydrophobicity of QPI E/S refers to the mass ratio of the enzyme to the substrate. Different lowercase letters indicate significant difference (P<0.05). The same as below"

Fig. 3

Effects of different enzyme-substrate ratios on the Th T fluorescence intensity (A) and morphology (B) of QPI aggregates"

Table 3

Effects of different enzyme-substrate ratios on the morphology of QPI fibrillar aggregates"

组别Group E/S 0.05% E/S 0.08% E/S 0.11% E/S 0.14%
长度 Length (nm) 225.98±80.44bc 312.20±94.24a 182.16±47.12c 247.82±87.34ab
直径 Diameter (nm) 10.13±2.27a 5.46±2.34c 7.87±1.50b 9.84±1.61ab

Fig. 4

Effect of different enzyme-substrate ratios on the appearance and microstructure of QPI gels Scale bar is 20 μm"

Fig. 5

Effects of different enzyme-substrate ratios on the texture of QPI gels"

Fig. 6

Effects of different enzyme-substrate ratios on the rheological properties of quinoa protein isolate"

Table 4

Effects of different enzyme-substrate ratios on the protein secondary structure of QPI gels"

酶底比
E/S (%)		 α-螺旋
α-helix (%)		 Β-折叠
β-sheet (%)		 β-转角
β-turn (%)		 无规则卷曲
Random coil (%)
对照 Control 13.74±2.59a 14.45±0.32cd 30.12±0.72a 37.45±0.67bc
0.05 12.39±0.50b 15.81±0.81b 26.95±1.84c 36.69±3.19c
0.08 13.04±0.26ab 16.85±1.39a 27.52±0.41c 39.17±0.45a
0.11 12.77±1.03ab 15.01±0.62c 28.81±1.08b 38.16±0.39ab
0.14 12.97±0.63ab 14.20±0.68d 28.93±0.84b 38.35±0.64ab

Fig. 7

Effects of different enzyme-substrate ratios on the protein secondary structure of QPI gels"

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